Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact

Abstract

Staphylococcus aureus (Sau) strains are a main cause of disease, including nosocomial infections which have been linked to the production of biofilms and the propagation of antibiotic resistance strains such as methicillin-resistant Staphylococcus aureus (MRSA). A previous study found that Streptococcus pneumoniae (Spn) strains kill planktonic cultures of Sau strains. In this work, we have further evaluated in detail the eradication of Sau biofilms and investigated ultrastructural interactions of the biofilmicidal effect. Spn strain D39, which produces the competence stimulating peptide 1 (CSP1), reduced Sau biofilms within 8 h of inoculation, while TIGR4, producing CSP2, eradicated Sau biofilms and planktonic cells within 4 h. Differences were not attributed to pherotypes as other Spn strains producing different pheromones eradicated Sau within 4 h. Experiments using Transwell devices, which physically separated both species growing in the same well, demonstrated that direct contact between Spn and Sau was required to efficiently eradicate Sau biofilms and biofilm-released planktonic cells. Physical contact-mediated killing of Sau was not related to production of hydrogen peroxide as an isogenic TIGR4ΔspxB mutant eradicated Sau bacteria within 4 h. Confocal micrographs confirmed eradication of Sau biofilms by TIGR4 and allowed us to visualize ultrastructural point of contacts between Sau and Spn. A time-course study further demonstrated spatial colocalization of Spn chains and Sau tetrads as early as 30 min post-inoculation (Pearson's coefficient >0.72). Finally, precolonized biofilms produced by Sau strain Newman, or MRSA strain USA300, were eradicated by mid-log phase cultures of washed TIGR4 bacteria within 2 h post-inoculation. In conclusion, Spn strains rapidly eradicate pre-colonized Sau aureus biofilms, including those formed by MRSA strains, by a mechanism(s) requiring bacterium-bacterium contact, but independent from the production of hydrogen peroxide.

Antimicrobial Activity of Clinically Isolated Bacterial Species Against Staphylococcus aureus

“…Finally, it is worth noting that some antagonistic interactions are more complex and can involve both contact-independent and dependent mechanisms. For example, initial physical contact between Streptococcus pneumoniae and S. aureus induces S. pneumoniae to generate and secrete hydrogen peroxide that can then kill S. aureus (Khan et al, 2016;Wu et al, 2019). Similarly, P. aeruginosa can physically senses S. aureus, which leads to global changes in transcription, resulting in the secretion of multiple compounds that have anti S. aureus activity (Korgaonkar et al, 2013;Filkins et al, 2015).…”

Interaction between Streptococcus pneumoniae and Staphylococcus aureus Generates·OH Radicals That Rapidly Kill Staphylococcus aureus Strains

“…The proposed mechanism involved the production of hydrogen peroxide (H 2 O 2 ) that was released by S. pneumoniae into the supernatant (20). This H 2 O 2 -mediated killing of S. aureus occurred within 6 h post-inoculation of S. pneumoniae, but it was inhibited in cocultures with catalase added; by incubating these cocultures in an anaerobic chamber; or by a mutation within the spxB gene, encoding the enzyme streptococcal pyruvate oxidase, which endogenously produces H 2 O 2 during conversion of acetylphosphate from pyruvate (19)(20)(21)(22)(23). Notably, SpxB accounts for ϳ85% of the membrane-permeable H 2 O 2 that is released by the bacteria into the supernatant (24,25).…”

Hydrogen peroxide production byStreptococcus pneumoniaeresults in alpha-hemolysis by oxidation of oxy-hemoglobin to met-hemoglobin

“…Oxy-hemoglobin (Fe +2 ) is autoxidized to met-hemoglobin (Fe +3 ), or oxidized by radicals such as hydrogen peroxide (17,18), inducing spectral changes, i.e., flattening the oxy-hemoglobin absorbance peaks. Spn produces and releases abundant hydrogen peroxide into the culture supernatant that intoxicate human cells (19), or that rapidly kills Staphylococcus aureus strains and other bacterial species (7,20). Hydrogen peroxide is a byproduct of the metabolism of two different enzymes, pyruvate oxidase (SpxB) and lactate dehydrogenase (LctO) (5).…”